#303 - A breakthrough in Alzheimer’s disease: the promising potential of klotho for brain health, cognitive decline, and as a therapeutic tool for Alzheimer's disease | Dena Dubal, M.D., Ph.D.

30:23When it's actually just a formality because the answer is so clear looking at the data, that's the really big aha moment. And I think I'd be safe in saying that that was the case here, wasn't it? You're right. We tend to be really conservative. We don't want to make an error. But having said that, you're totally right. I mean, I can imagine the graphs now. And there were non -overlapping data points. I didn't need to run the repeated measures between subjects and NOVA to really know that it was statistically significant. and it was that type of a finding. So it's a point well made. Sometimes experiments will show you those differences and then the next time you do it, they don't.

31:09Someone who runs the water maze is wearing a new cologne and the mice get stressed out. They don't always repeat and are not always replicable. This is a case that it was amazing, but was it going to replicate? Was it gonna be true in different cognitive tasks? what would happen in aging mouths, like all of these questions opened up, and we iterated, and we did experiment after experiment, and the data held strong in mice. That clotho overexpression really enhanced their cognition, in young mice, in aging mice, in mice that modeled Alzheimer's disease, and also in my publication that's in peer review now, but also in my set model Parkinson's disease that overexpression of clotho enhanced cognition.

32:03It is really a remarkable finding. I think it's maybe one of the most important findings in my professional career. I would go one step further. I feel like it's one of the most important findings in brain health period. I mean, on some levels, It's a bit of a mystery to me why this isn't known by everyone. It's part of why I'm really, really happy to be sitting down with you and why I've wanted to sit down with you for the better part of six months. And I know that between my schedule and yours, it took a minute to get us on the schedule. But this is such a big deal. I also want to reiterate something you said a moment ago and I say this and we'll be linking to all of these studies so people can see the data for themselves.

32:44but the reproducibility coupled with the magnitude of the effect, so the consistent, enormous magnitude of difference across studies, across labs, across investigators, that signals something is going on here. Again, not to bring up rapamycin, again, which people hear me talk about a lot, but people say, why are you so confident in rapamycin's genocidal, zero protective effects. The magnitude of benefit is enormous, and it doesn't seem to matter who does the study. If it's done in this part of the country, or this part of the world, or with this animal, or with that animal, it always seems to work.

33:24Now, that doesn't guarantee it's going to work in humans. I'd be the first to admit that. But boy, I feel a lot better when it works in every model system virtually every time and when it passes the eyeball test, you don't need the error bars and the p -values. So let's talk a little bit about before we go to primates and ultimately to the epidemiology and humans. Let's talk about mechanism of action. Tell me what you think is happening with this soluble, clothoprotein. I assume it's crossing the blood brain barrier or it wouldn't be having this effect. Has that been easily demonstrated? It is a winding story, Peter.

34:07And one that's exciting and has led us to very unanticipated findings. I first wanted to mention, as we think about how different interventions may converge upon the same biology or relate to the same biology, it is interesting that mice treated with rapamycin have increased clothal levels. So, rapamycin, either directly or indirectly, will increase clothal levels, just thinking about the convergence of biology. Now, how does clothal enhance cognition and a young, aging, and disease brain? And that immediately became a major question for my lab and many other labs. And the very first thing that we had turned to was a component of NMDA receptors.

34:54So NMDA receptors are really key in connections between neurons. And when they're stimulated, they let in calcium into a neuron, and they allow essentially a potentiation of a neuron for connections to form, for functional connections to form, and really for the neural substrate of memory to form. So NMDA receptors are really key, and there's a component of NMDA receptors called glue and tubing. It's a subunit. I'm really going tiny and molecular here, but it's very important because I had remembered that there was something called a duge house or mouse, affectionately called a duge house or mouse.

35:36We're kind of dating ourselves here because there's going to be a lot of listeners who don't know who duge house or is, but suffice it to say this was a prodigious mouse. Yes. So it was a mouse that was smarter. There are very few model systems where you can actually pivot the system to make an engineer mouse to be smarter. There are very, very few things. Clothos, one of, so this glue end to be overexpressing mouse or the doogie house or mouse showed a very similar phenotype to the Clotho overexpressing mouse. When I looked at the behavioral data of the glue end to be overexpressing mouse in the very same test, it was performing very well, just like the Clotho overexpressing mouse.

36:18So I just wondered whether clotho was acting through glue and tube to enhance cognition. That was the hypothesis. In this case, the hypothesis, we gathered data that supported the hypothesis. There wasn't a big surprise in that overexpressing clotho caused an increase in glue and tube at the the naps at the area where brain cells connect and lead to a more efficient connection and memory formation synaptic plasticity at the level of the neural cell. So this glue end to be was really important when we blocked glue end to be with multiple pharmacologic inhibitors, we abrogated clothos ability to enhance cognition.

37:06So that was one clue, but here's the major head scratcher. So, Peter, clotho is expressed and the kidney is also expressed in the brain, secreted through the cord plexus, which also makes the fluid for the brain, that the brain sits in. But clotho does not cross the blood brain barrier. We have looked, others have looked through auto radiography, through IP and Western Blot, we've looked through many immunohistochemistry, we've looked through many different methods and can see very clearly that it doesn't cross into the brain. However, when we give a shot of cloth though, to mice, to monkeys, in their arm, or their belly, it doesn't matter if we give them a shot of cloth though, within four hours there is cognitive enhancement, and that cognitive enhancement lasts for at least a couple of weeks.

37:58So how is it that giving peripheral cloth though is enhancing brain functions if it's not actually crossing into the brain. That's been a major question that we and others have been working on. And we have some clues that led us in unexpected places. I'm happy to talk about one of them if this is a good... I am all ears. To me, this is... I mean, I didn't want to get to this point yet, but this is the natural time to talk about this. And then I think we should step back and talk about some other things. But yes, to me this is the jugular question, Dina. Why is it that when we inject a monkey peripherally, with clotho, it spends the next 14 to 19 days in a state of cognitive superiority?

38:48And that clotho isn't crossing the blood brain barrier. This is to me the jugular question. You're right. And we have been working on this. I think I'll step back and say, it's actually amazing that you give something peripherally. It has a central action. It's not crossing into the brain. And it's something physiologic that our bodies are used to, that we have seen high levels of upon birth and development. It does not have known side effects at physiologic levels that have been observed yet. It's very remarkable. So what is it doing? We hypothesize. So I'll just say we don't know yet. This is an area of fervent investigation for us and others.

39:35But we have one clue in a study that we published recently in Nature Aging. And this is what we did. We started with the premise that maybe clotho was doing something in the blood that then would send a messenger into the brain. So maybe there was this messenger of clotho that was actually going into the brain when you gave it peripherally. And so we did a very simple experiment. We injected mice with clotho and four hours later at the time of cognitive enhancement. We did an agnostic profiling of the proteins in their plasma in their blood and what we found was sort of unimaginable. It was unimaginable in that we found that all of these platelet factors were increased with clothoinjection in the blood.

40:27And Peter, I have to be honest when I saw this agnostic data set, I turned to my postdoc and I said, I'm really not interested in this. I am not interested in looking at platelets. They are involved in wound healing, their coagulation factors. This is weird. I had a postdoc, Kena Park, who was very persistent and said, let me try. Let me just study this for a little bit. I said, go for it. I could see your passion. One question, Dean, just to ask, in parallel, did you do the same multi -omic observation of the CSF in the mice in parallel to see if there was something new that was showing up in the CSF to parallel the platelet factors in the plasma?

41:16Yes, we did. We haven't published this yet, but we did. In the same mice, we took their blood and we took their CSF. And what we found is that cloth will increase these platelet factors. It was totally bizarre. I didn't believe it. I tend not to believe data. Like, need to see things over and over again. Need to see functional studies. I need to see it matters. I tend not to believe data, but there it was. She repeated the findings in many different ways, but ultimately what she did was a series of studies demonstrating that this biology is real. The cloth is actually activating platelets very modestly and then when a platelet is activated, so let's first just step back what is a platelet?

42:03A platelet is an A nuclear cell in our blood and it contains little compartments of bioactive chemicals, chemokines. And when a platelet is stimulated or activated, it actually releases these factors. And it does so in a context -dependent way. So when there is a cut or a wound, there is a huge activation of platelets and they release all sorts of factors that help with clotting and help with the wound healing. So that's what they're traditionally known for. But it turns out, Peter, that when we exercise, our platelets are activated, and they're releasing factors that travel into the brain. I mean, who would have thought that platelets could be messengers of brain health and could take center stage as a messenger of brain health?

42:57So when we exercise, this was found by an Australian group led by Tara Walker, that when we exercise platelets are activated and certain platelet factors, one of them being platelet factor four, is released and travels to the brain and actually causes neurogenesis or the production of new neurons in the brain. That was very new data. And with that data in mind, I felt too like maybe there's something to this cloth will platelet connection. Maybe there's something to it. So, Kena isolated platelets. She put clotho on them. They released PF4, this platelet factor 4, the same one that the Australian group had shown.

43:43And then she gave this platelet factor 4 to mice, young, aging mice. She gave it to them as a shot in the belly, just like we have given clotho. And she found that that platelet factor for enhanced cognition and a young mouse and an aging mouse and it reversed cognitive deficits and it was totally remarkable. This is where it gets weirder and wilder. So I go to my colleague and close friends, solvalata here at UCSF. He's the young blood and old brain scientist. So he's really built a really nice body of experiments and literature showing that if you give young blood to an old mouse that rejuvenates their brain.

44:26So, Saul is studying brain rejuvenation through blood, and I'm having coffee with him. I said, Saul, we've found something so interesting. I have to tell you about it. And I go on to tell him about P .F .4. Klauffo stimulates platelets. Platelets release P .F .4. P .F .4 enhances the brain. And he said, well, he was silent for a second. And he said, Dina, we found the same thing with young blood, and that when we give young blood to old mice, that young blood is enriched for platelet factor 4, which declines in aging, and then we give platelet factor 4, we rejuvenate the old brain. And if you think that's a remarkable convergence of biology, wait till I tell you that Tara Walker and Australia had found the same thing with exercise, and this is all at the same time that exercise was increasing PFOA and that when she gave PFOA to hold mice it enhanced their cognition.

45:30And so all of us who are close friends and colleagues now have an incredible convergence of biology, reclothal, young blood and exercise for activating platelets, releasing PFOA, PFOA is enhancing the brain. And as an aside, like the biology is just amazing, each of us had our own unique approaches and unique way of digging into the biology. But a practical question, Peter, was like, what are we going to do? Like, are we competitors now? Or are we going to hold hands and go through the publication process together? Are you going to screw me? Am I going to screw you? How is this going to come out.

46:13And what we did is I think a really nice example and model in biology or re -held hands. And we said, let's just stick together. We went to editors. We went through a couple of years of revisions and reviews. But at the end of the day, all three papers came out and the Nature Family of Journals on the same day. It made for an impactful splash. And it also just highlighted the convergence of the biology, the reproducibility of the biology. Again, this will never happen again in my lifetime, but this was amazing and a complete surprise that platelet factors could play a role here. So let's talk a little bit more about this.

46:55So platelet factor 4 is increased by all of these three things. Do we believe that it's platelet factor 4 that is directly acting on glue and to be, then, and if so, what do you think is happening at the level of substrate and receptor? That's where we went next with the biology and the experiments. And so one question was, does platelet -backed for actually cross into the brain? And we demonstrated that it does. We gave it peripherally. We immediately looked into the brain and saw that it was ending up near neural cells or even within neural cells. So it's crossing into the brain. And then does it act directly into the brain?

47:41Was the next question. And what one of my lab members did is take hippocampal slices of the hippocampus is the area of the brain that is really executing the learning and memory and is targeted by aging and diseases of aging. So he took hippocampus And then he put platelet factor 4 on it and saw immediately within seconds, actually, there was a, well, I don't know within seconds, but quite immediately, maybe seconds, maybe minutes. There was a change in the membrane potential and more calcium was being let in. And we thought maybe this would be glue N2B acting. and so we put on gluentubi inhibitors and platelet factor 4 no longer potentiated the synapse or enhanced the neural function.

48:35So we do know that platelet factor 4, like clotho, is working through gluentubi. And maybe clotho is working through platelet factor 4 to change gluentubi. And sorry, has that same experiment been done, Dina, to look at the clotho that is derived from the Choride Plexus to say, when you dump clotho directly onto glue N2B, you see an influx of calcium. When you block glue N2B, you abrogate the effect as well. So you now have two things, platelet factor four and clotho that can independently do the same thing. Is that a correct statement? We haven't done that experiment yet. We've done transgenic overexpression in the brain, and we've done clothopriferally.

49:23We haven't dumped clothopo itself onto the hippocampus to see whether there can be a direct effect. Other groups have overexpressed clothopospecifically in the brain and seen cognitive enhancement, but I haven't seen studies seeing whether clothopo is actually directly influencing Luan to We know that it indirectly influences gluten to be. We know that we can give it peripherally and it'll increase synaptic plasticity through gluten to be. But we don't know whether it can actually directly do that to it may, based on other people's studies it may. We don't know, but here's something else that was actually quite disappointing.

50:05But the biology is the biology. We then ask the question, Disclotho rely on PFO to enhance cognition. It's PFO required for cloth -omediated cognitive enhancement. And to do that, we generated a colony of mice with PFO knockout. They just don't have PFO. And then we gave them cloth -o versus vehicle, always blinded, always randomized, through a series of cognitive tests, water maze, large Y maze, one maze after another, because you like to see the same effect reproduce. And what we found was that cloth will continue to enhance cognition, even in the absence of platelet factor war. To the same extent, Dina.

50:54To the same extent. You might think there would have been a diminution, but to the same extent. It just tells us that there's more than one factor. It has to be such important pathway, and it is so conserved that there is no way biology is going to let one thing be the messenger. I'm making all this up, of course, but hypothesizing that if Klautho is so important, it can't be limited to one messenger. If it gets to the gate and it can't get through, it has to have multiple messengers that it can deliver the message to PF4 being one of them, but clearly this experiment would suggest that there's at least one other messenger, right?

51:33Right. That's the interpretation and conclusion because PFO is sufficient to recapitulate proper mediated cognitive enhancement, but not necessary. And so when you go back and look at the first experiment you did, the unbiased, was that just a proteomic assessment or did you look at all omics? How broadly did you sample the serum way back? And was there something else you missed because the PF4 was such a big signal? Was there a smaller signal that was there perhaps as well? There were many signals. There are many other platelet factors, there are many other proteins. We are systematically marching through those.

52:14We're marching through not just proteins, but metabolites. We are also asking in a cell type specific way through a system called turbo ID that labels the protein as it's secreted from the liver, the kidney, the heart, from lymphocytes. It's a really unique and elegant genetic manipulation that does a biotin label once the protein is secreted, let's say from the liver. And we're asking when we inject clotho, what gets secreted out of the liver and into the plasma. So we're doing like higher resolution studies to understand and how clotho really changes the systemic circulation in a cell type specific way, and then asking which one of those factors that comes from the kidney, the liver, the lymphocytes, the heart, which one of those factors is necessary and required for clotho -mediated cognitive enhancement.

53:14And the answer at the end of the day may be what you alluded to that there may be many factors that have overlapping functions of cognitive enhancement. Maybe they work together, maybe they work independently, but you're right, if this is a very important to biologic function, it really shouldn't rely on just one factor. I think that's a really important point to underline. Tell me a little bit more about this assay, the in vitro assay, that you were able to basically sprinkle on PF4, sprinkle on clotho to the glue N2B subparticle and witness the massive influx of calcium. It seems to me that if that assay is reasonable, it could also become a great screening tool to identify rather than having to do the experiments to see if you can enhance cognition, at least do your first screen there.

54:06Now you run the risk that if clotho is working through secondary mechanisms, you'll miss it. But at least as a first screen in as much as Klaatho's working through an MDA via Glue and 2B, let's screen a whole bunch of molecules on it, look for the activation, and then go back and search for those in a biased way in our serum sample, and sort of triangulate back and forth like that. I love it. Let's do it. And we are doing it and we're doing it with synaptic plasticity as a measure as a substrate of cognition. We're also doing it in vitro at the cellular level using live cell imaging where we can isolate neurons from the brain from a mouse brain and then they grow beautifully on a dish.

54:57They connect with each other and they survive for weeks on end actually. And you can do live cell imaging to see whether if you put cloth on. I have a graduate student doing this right now, Barbara Shariva, who is putting cloth on to neurons and these other factors that we're seeing from the omics onto neurons and seeing whether there is an increase in the neurite outgrowth and the connections, the physical connections between neurons. Again, this isn't cognition itself, but it's a substrate of cognition. It may be a distant biomarker for it, but it's a really smart way to, when you're asking a question of this many proteins, which ones are important.

55:44It's a way to screen. So with synaptic plasticity and with neurite connections, with the outgrowth of the neurons connections. So, all of this makes a lot of sense for a mouse model of Alzheimer's disease where the primary deficit is a cognitive deficit that seems disproportionately focused in memory. But you mentioned before I distracted us down this rabbit hole that you are also seeing positive signals in a mouse model of Parkinson's disease. Now, are you seeing them in the mild cognitive impairment that can accompany Parkinson's disease? or are you seeing it in the movement disorder? Are you seeing an improvement in the primary issue associated with Parkinson's disease?

56:29Part of this is published. Part of this is in the peer review process, but it's really, really exciting to share. So these are the experiments. We took mice that overexpress alpha -cinuclein, which is a pathogenic player in Parkinson's disease. disease, it disrupts the synapse and has a cause to role in Parkinson's disease because we know that people with mutations in causing overexpression of alpha -synuclein will develop Parkinson's disease. It's sort of like the equivalent of APP in people with Alzheimer's disease where you have this, it's not necessarily the dominant driver of the disease, but it's clearly playing a causal role based on these mutation studies.

57:14That's right. And then the mice that I've mentioned that we've used for our Alzheimer's or mouse hemorrhage studies were APP mutant overexpressors just to pedal back to that. So we took these Parkinson's model mice and they have both motor deficits, meaning that they walk across a balance beam and they'll fall if they're put on a spinning rod, they're cognitive difficulties, which are not as severe as the Alzheimer's model mice, but they do have cognitive difficulties and their ability to map a spatial environment and to hold memory in their mind with working memory. So they have these deficits.

58:00We did two things. We injected them with cloth and remarkably saw that clothotreatment improved their cognition. It didn't normalize, but it improved if I'm thinking about the data correctly by maybe 70 % or so, their cognitive abilities. It's a nearly normalized, their cognitive functions. It didn't do anything to their motor functions. So they continued to have motor dysfunction, and clothot did not help that. That was also true with the transgenic over -expression of cloth, though all over the body and brain for a lifetime, that awarded the cognitive deficits induced by Parkinson's toxicity, but it did nothing for the motor problems.

58:50And I've spoken to my Parkinson's colleagues about this. There's a lot of interest in potentially using cloth, who is a treatment for Parkinson's disease, because they tell me in our clinics, you know, we can treat the tremor and the rigidity, but people complain over and over again, consistently about the cognitive deficits that we now know are part of Parkinson's disease, not just later in the disease, but even as part of the disease. And those deficits specifically are problems with executive function, which is the ability to focus, to shift attention, to make certain judgments, to think quickly, and it is mediated by the prefrontal cortex, an area that became important, we looked at human studies.

59:45So the bottom line was that in mice, clotheau really enhanced, again, cognition, but didn't do anything for motor functions. Is there a mouse model that fits between the Alzheimer's model and the Parkinson's model that is more akin to a Louis body dementia model where it has some of that alpha -synucleon pathology, but also has a much more significant cognitive component? There are so many mouse models, Peter. I think that a good mouse model for Louis body would be alpha -synucleon and maybe more in the brainstem and the hippocampus and cortex. I bet there is one, but I'm not clear. But a reasonable hypothesis would be, again, that it would probably provide some relief of the cognitive symptoms, though not necessarily the movement symptoms.

1:00:40That's right. So that's what we're seeing consistently. One of my friends and colleagues had an interesting analogy of thinking about clotho as a helmet for neurons that whatever came crashing the cells way, whether it was Alpacenucleon, Tau, Amaloid Beta, aging stresses that the neuron was protected. It remained resilient against multiple toxicities. And the effect of cloth, though, is really in cognition itself in hippocampal and frontal cortical circuits that these neurons and glia and other cell types are really protected against multiple toxicities. If we want to jump into clinical trials, I think this is the time to really move cloth out toward human clinical trials.

1:01:27Wouldn't it be amazing if we had a cocktail for Alzheimer's disease? In addition to Lecana Mab or Denanamaab that's removing the amyloid beta from the brain, what about adding something like clotho that can really help shore up the functions of the cells because we know that Alzheimer's disease is a multi -proteanopathy. It's not just one protein that's causing the disease. We need factors in our treatment. We need cocktails that can really resist multiple protein toxicity. So I just have this dream that people might be able someday to benefit from clotho. This factor that naturally circulates in our body that helps with longevity, that helps with other organ systems, and enhances the brain.

1:02:19As we know from our monkey studies, for weeks at a time, at least a couple of weeks if not more, there is funding for a certain amount of time, but it has a long -acting action. We need to put helmets around the neurons to really stave off multiple toxicities. And this is clotho. This is what clotho does. is the new job of the Greek fate that spins the thread of life. It's to protect our brains. Yeah, you're preaching to the not just the choir, but the fully converted and obviously in my other activities, this is something that a lot of my energy goes into. I want to come back to a couple other questions on the mouth study that are going to factor into, I think, when we start to talk about the humans more.

1:03:06Has the following experiment been done where you take a mouse that is genetically susceptible to Alzheimer's disease? So the equivalent of your APP mouse or something like that. And you take a control group, maybe just for my own knowledge, if you took an APP mouse at what point in its life, how many months old before it starts to develop a clinical disease? So maybe two questions. At what point would it start to be accumulating amyloid in the CSF? At how many months of age and then how many age when it starts to show cognitive impairment? What are those two numbers roughly? It depends on the mouse model and there are so many mouse models of Alzheimer's, including APP models.

1:03:50The one that we've used consistently is called the J20 model. It expresses human APP, which produces amyloid beta in mutant forms. And it's a more aggressive model, Peter, and it causes synaptic loss that connection between cells that really disrupts that connection between cells really early before three months. And then it starts producing cognitive deficits at around three to four months. And that's the J10. The J20 model. J20. Okay. So here's my question, Dean, has the experiment been done where you give those mice cloth though starting at birth at a high dose and does it delay the onset of the inevitability?

1:04:40We know that through overexpression, throughout the brain and body, those mice,

1:04:55those on said there are others that have done human population clinical studies. I'm going to make sure I understand what you just said. You're saying if you take the J20, the APP mutant and you also cause a mutation that overexpresses cloth though, are you saying that you've done that in the same mouse? Yes. Tell me about that mouse's lifespan. Is it normal and is it cognitively normal? Or does it still get Alzheimer's disease? That mouse that has the APP mutation and over -expresses cloth though by about three to four -fold, that mouse will live much longer, number one, so it extends its lifespan.

1:05:38Number two, it will normalize its cognition. Across its lifespan? Across its lifespan. At three months, seven months, eight months, yes, it normalizes its cognition. It's very remarkable. And then when you look in the brain, Peter, when you look at levels of amyloid beta and of tau, they're not different from the mice without high levels of classo. That is unbelievable. In other words, their brains are still riddled with the Alzheimer's toxins, but they've been able to really they've thwart the effects of those toxins because they show normal cognition. And if you look at their synapses, this work was done in collaboration with Leonard Mookie and Elise or Mauslia, who's at the NIH now, when Mauslia looked at the synapses and these mice, he saw that the synapses were all preserved.

1:06:41So again, this analogy where Klauffo is really providing a helmet around each neuron, It really allowed the synapses to be preserved, but there was a whole bunch of amyloid and Tao still there. And that is resilience. These toxins are present. They won't necessarily go away with cloth, though, at least in mice. The story may be different in humans, but cloth, though, provided resilience and thwarted the toxicities of Alzheimer's disease. Okay, so I'm going to plant the seed with you, which will come back to. But the reason I'm asking this question, Dina, is I do believe that at some point we will have cloth though as a drug.

1:07:25I think it's going to come to market. We're absolutely going to be injecting people with cloth though. Where we'll go is do we believe this is a drug we will only want to give to people once they have MCI for the listener, right? So early early early stages or do we believe that we'll take anybody who's susceptible and just be giving them cloth though even before there is a demonstrated disease risk. So that's where I want to go with this is kind of thinking three or four moves ahead on the chess board. Now let's go back and talk about the primates because we've talked about this incredible body of literature in mice and again, I think it's reasonable to be circumspect around mice literature But again, when you start to talk about the volume of literature here and the breadth of mouse models, and the number of investigators that consistently find the same results, and the magnitude of the results, again, it passes the eyeball test, you don't need to do ANOVA tables all day long to get the answer and squeak at it.

1:08:30This looks incredible. But it would be nice if we had a more convincing model. So let's talk about the single best thing we can ever do before we get to humans and that's to look at primates. So let's talk a little bit about primate brains and the body of literature around cloth in that brain. This is an area that I wanted so much to get into. I don't work with monkeys. I think it's a very, very specialized field to test cognition in monkeys. And so I'll just take us back a long time ago, maybe eight years ago, when I really wanted to know, Peter, whether cloth can enhance a more complex brain than a mouse brain.

1:09:16And like you said, mice are really important in scientific discovery, but there are so many examples of what worked in mice, and cured mouse -hymers disease, failed in humans. And that's the valley of death. Works in mice doesn't translate to humans. It's not true for everything, and mice have been very, very important for fundamental scientific discovery and for medicines. But I didn't want to spend my career on something that was not going to be relevant to humans. And that might not be that big and important thing that Stephen Hauser challenged me on when I was a resident. I was pitching this one -a -test cloppolymphimates all across Silicon Valley to biotech and and people were curious and enthusiastic about it, but one individual really got this, Ned David, who had started a company unity by a technology to attack aging.

1:10:17And we joined forces. They fundraised, they found really great collaborators at Yale and really made this happen to test cloth and cognitive enhancement in a brain like ours. When people would tell me if it doesn't work, it's going to kill your research program. And I would say let it die then. I don't want to spend my career on something that's not important. If it's done well, we'll know. And so this collaboration between biotech, me, and Yale was one in which a lot of thought, a lot of money went into the design and the execution. So why non -human primates? Rhesus macaques are incredible organisms.

1:11:04They have very complex brains. They are more genetically diverse than humans. And that genetic diversity is something that often trumps effects. Can often really influence whether an effect is able to survive genetic diversity or not. So having a lot of genetic diversity is very important to really challenge and test the biology. Mice are in bread. They all have the same genetics just to say. So the recessic macaques are incredibly genetically diverse, more than humans. They have a functional complexity that's very similar to humans in terms of the even more of the neural circuits, the hippocampus, the prefrontal cortex, and they have an anatomic complexity that's pretty similar to us.

1:11:53So by testing cloth, though, and non -human primates, the idea was to jump over this valley of death. If it worked in primates, let's look toward humans. If not, let's do something else. I want to pause there for a second, Dina. I mean, I think, obviously, the listener can imagine where this story is going, but that's a huge burn your ship's moment. I want to just spend a minute probing that a little bit more and you were a wildly successful investigator before this. I don't think it's an obvious step that you would take to push that much further because let's be clear. If clotho had failed miserably in primates, how many PhDs in postdocs and students do you have in your lab?

1:12:42We're a group of around fluctuates between 7 to 11. Yeah, and obviously you probably have a very well -oiled machine at generating R1s and all sorts of grants. It's not easy to start all over again if all of that dries up, right? Well, that's why it was so risky and some people just go to a human trial. You can do biomarkers, but this is risky. And it is risky if it's not done well, but it's super informative even if it's negative. If it's good negative data and not because the experiments were done in a sloppy way, it's good negative data, then it really tells you something. And we have other projects on longevity.

1:13:27We study why women live longer than men. What does a second X chromosome have to do with it? And there are other longevity factors. So it was really important to push forward and to push forward in a rigorous way, and also with the knowledge that the results could really change the direction of what we're doing. But we have times in our lives where we reflect on what we're doing, how we're spending our time, is it with people that we appreciate, is it doing things that are meaningful, and it may be deeply philosophical, but from day to day, I really want to be doing something that I a lot of faith in and that potentially has meaning for the human condition to improve human health.

1:14:14And if we've done a really great experiment that's negative and there's really reason to believe that this isn't going to go forward then, that's fine. We will mourn it and we will still move forward with something else. But we just don't have lifetimes to devote to 100 different scientific big projects. So we have to be very choosy and I really, really wanted to make sure that this is something really worth putting. Time, energy, resource, passion, constant thought, training, fundraise. I think the reason I'm pointing this out, Deena, is I want to publicly commend you for something that honestly I don't think most scientists would be willing to do.

1:14:55And I wouldn't even hazard a guess at the fraction, but I think there are a lot of scientists who have lost sight of what you just said and they think the job is doing science. But it's not. The job is knowledge creation. The job is knowledge creation for the purpose of making lives better. And those are very different things. They overlap. The former is a process to the latter, but it's very easy. and it's very easy to lose sight of that in the weeds. So I only call it out to said, I don't think everybody would have come to the same conclusion you did. So with that said, let's talk about how you and your colleagues at Yale and basically thought of the right question to ask and then the right design of an experiment to ask the right question.

1:15:49First, I just want to say thank you and that is very well said. Monkeys undergo cognitive decline in a very similar parallel fashion to humans. They have synaptic loss, the connection between neurons with aging as do humans do. And the circuits that are affected in aging are really similar to the human circuits. So they are a hippocampal frontal circuits, the hippocampus and the frontal lobes, and aging will preferentially target working memory and also spatial memory and other types of memory. But this working memory, holding something in your mind, you go to the refrigerator, you open the door and you think, why did I come here?

1:16:38What was holding that immediate memory in your mind is something that aging really targets. and monkeys undergo this very similar cognitive decline to humans. And there are ways to test monkeys that interrogate those circuits. In this case, a spatial delayed task was used. Grammoliums and Stacey Kastner conducted the studies. And clotho actually preferentially targets those circuits that aging erodes. So here's a model system. It's genetically, anatomically, functionally complex. It undergoes cognitive aging, parallel to humans. And there are really well developed tests by these exceptional scientists who are very experienced and adapted conducting these tests.

1:17:28So it was a really excellent set up to ask Ken Clotho, enhance cognition in these aging monkeys. What was done is something called a spatial delayed response. And the monkeys presented with a bin of multiple wells. And sometimes there's a few wells and sometimes there's a lot of wells. And it's harder when there's a lot of wells to find a treat. So a treat is placed in a well and they can see you place the treat and then a screen drops and then all the wells are covered. You cannot see where the treat is and then the blind is opened and they are tested where to choose because they're basing this on spatial memory and on working memory where that treat was.

1:18:17Again, it's a little easier if there's only three wells. It's easier to choose one out of three and get it correct. It's harder when there's nine wells to remember spatial and working memory where that treat was. It was done very rigorously. I must say the study was largely blinded and it was done in a way in which Injection of clotho wouldn't interfere with causing stress and memory so that everyone got a baseline They then got a vehicle and then they got vehicle or clotho It's a really well -designed study and just for folks listening vehicle is placebo so that they understand what we mean by that.

1:18:55Thank you. So the monkeys that got a cloth of treatment, again largely blinded, performed better than the ones that got vehicle treatment. Sorry, Dana. How did you guys decide how to raise the dose? So first of all, when you obviously did all the mice experiments, you're dosing like you probably do based on a certain number of milligrams per kilogram or milligrams per gram of animal. Well, what did you use to dose escalate that? Did you just assume the same dose per unit body weight or did you make other adjustments? Well, we went with two things in mind. One is we really wanted to stay at least with one dose in a physiologic range.

1:19:40So with something that the body has been exposed to and has seen over its lifetime. And so in one dose, we wanted to go somewhere up to like, it may be four to five. Yeah, yep. You're still just below what they would have been born at. Right. A very useful level of cloth oats, a rejuvenating dose. And then the other doses were higher. And were meant to test, could you really push the system to improve cognition even more than what is observed with that physiologic dose. By the way, that physiologic dose, that sort of natural dose, is what we have always used in mice. I should say we've also given mice huge, huge doses that were way beyond what they would ever see or used to, and it still enhanced cognition in mice.

1:20:33And that's going to be different than the monkey story. By the way, when you gave this super -physiologic doses to mice, So presumably 10 fold and beyond. Did you run into any problems? For example, did the mice ever develop antibodies to the protein? Did anything else arise that would suggest that more is not always better? We didn't see signals in mice, but having said that, we didn't systematically study whether doses of 100 micrograms per kilogram was doing something different. We didn't observe differences in their normal behaviors or like in their basic blood work, but it worked. It didn't work better, so I should say like those super low doses were just as well as the super high doses in mice.

1:21:25And super low doses mean well below 3x normal. So if we just call 6x the peak physiologic dose, what was the minimum effective dose in mice relative to that? more than five times less, a very, very small dose that still worked. I'm thinking if we gave my 10 mA per kilogram, I'm remembering something like 0 .5 or 1 enhanced cognition. E2 micrames enhance cognition, so very, very low. Just a touch more, just a boost enhanced cognition. Our strategy was to stay within a physiologic dose and make sure that that was represented in the monkey studies. I mean, I really thought about this, consulted with people that have taken drugs to market.

1:22:16Tom Boone is one of them and was very instructive and taught me a lot in terms of thinking about relevantly dosing in monkeys. And then there were higher doses that created much higher doses and the body has seen, which is maybe 10 times higher and beyond. 10, maybe 20 times higher and beyond. And what we found is that the low physiologic, that natural dose of clotho, enhanced cognition in the monkeys. And it did so within four hours. And then that cognition stayed better. Their ability to think, remember, stayed better for at least three weeks, 14 to 21 days, and somewhere even tested out to a month.

1:23:02I saw 28 days. Yeah. And then at some point the study had to be stopped. It's a million dollars and more and so, but it was remarkable that one subcute dose like a shot of ozempic would be given a subcute dose that was low physiologic had an immediate effect on cognitive enhancement that lasted for a very long time. Now, there are a few different types of tests done. One was for normal memory load and one was for high memory load. And the high memory load was a test with a lot of different bins where they had to remember which one among seven or eight wells was the treat hidden in. It's just more taxing with more choices to remember.

1:23:49And they did even better in that study of high memory load. With a low dose. With the low dose. So the effect was particularly pronounced. They were particularly smarter when the task became harder. And again, why this task, why monkeys, why cloth, though, it all came together because cloth works on these circuits that are tested in the monkeys that decline with aging and with neurodegenerative diseases like Alzheimer's and Parkinson's. So what about the high dose? Right. So the high dose did not work. The high doses that gave the monkey's clotho way beyond what they've seen in their lifetime, they didn't harm, they did not impair cognition, but they didn't help cognition.

1:24:39And it's not too much of a stretch to think that too much of something that does multiple things in the body and maybe multiple pathways that could just create an imbalance that doesn't support cognitive function. Again, it wasn't harmful with the measures that we tested, but it didn't help to take so much more than the body has seen and is used to. And that again was different from the mice. So the mice, and this may be a big difference between mice and a more complex brain with the non -human primate. And mice, we saw continued cognitive enhancement, and in monkeys, we really got a window into before going into clinical trials that if we're giving cloth, though, probably should be thinking about a specific therapeutic window, one that the body knows, one of the body is used to.

1:25:31So, Tina, why do you think the effect lasts for so long? This is also a little counterintuitive when you consider the hourly variation of clotho naturally when you consider the transient effects potentially of things like exercise where you see these large boosts over a short period of time following about of exercise. But it seems hard to imagine that the benefits of an hour of exercise persist three weeks later, the way a subcutaneous injection of clotho did. So what do you think accounts for that? I don't know the answer, but I'll speculate. First I'll just say it's remarkable, isn't it? It's particularly remarkable as we imagine therapeutics in that maybe this is the type of treatment of rejuvenation that could be administered maybe once a month, once every three months as a shot in the arm, for example.

1:26:38So I think it has really important therapeutic implications. Mechanistically, this means that Klaafo has not just an acute effect of immediately enhancing NMDA receptor functions, synaptic plasticity, cognitive function at four hours, but it has an organizational effect. It's doing something to, in the longer term, really help to, for example, remodel a synapse. So I would imagine that, for example, with glue N2B being traffic to the synapse and to promote better functions, that those sort of synaptic organizational effects are happening for and staying put for a longer period of time. I think the biology of this organizational effect of cloth, though, has yet to be discovered, but it's something that's happening at the synapse.

1:27:37I have a very strong sense of something that's happening organizationally, structurally at the synapse. As much as I want to go deeper into that and talk about the conformational changes that could be occurring, we'll have to save that for another discussion because where we have to really go next is what do we think about in humans? And what evidence do we have, because we do have some really interesting evidence, even absent a single experiment, that everything we've talked about so far might indeed also be relevant to the species of interest. And no disrespect to the mice and the macaques, but there aren't too many people listening to this whose mind isn't already wondering, okay, enough about the animals already.

1:28:25Tell me, is this going to make a difference for my mom or for my dad who are in the early stages of dementia, is this going to make a difference for me because of my risk factors, even though it's 20 years from now? And so tell me a little bit about a particular SNP associated with the clothogene called KLVS and what its significance is to this story. None of this matters, Peter, if it doesn't have potential to help the human condition, it just doesn't matter. It's interesting and but the big and important is if it's relevant and may work in humans. I don't know whether we've done anything big until we test Klauvo in humans.

1:29:13So with that said, back in the early days, and I think it was 2012 or so when these are 2011, 2012, when these first Klauvo studies we were doing them in mice and discovering these cognitive enhancing effects. I went to, and again, I'm a physician scientist. I always have humans in mind. I have my patients in mind, Alzheimer's disease, 50 million people around the world. This is going to triple by 2050. Cognitive decline is our biggest biomedical challenge. Always have humans in mind. I went to my friend and colleague Jennifer Yokoyama, at the Memorane Aging Center here at UCSF, and I told her that there was this genetic variant of cloth, though, that had been found.

1:29:59We can talk more about what that is. But was there a way, because she's a geneticist, and she and others at the Memory and Aging Center, Joel Kramer, Bruce Miller, have built this incredible population of individuals and patients, Alzheimer's disease with normal aging, with front -to -temporal dimension, they really carefully characterize them. They're genetics, they're blood biomarkers, etc. So I went to Jennifer and I said, Is there any way we can know whether this genetic variant of cloth O -K -L -V -S has any association with cognition? Because that would mean that this cloth O is important to brain.

1:30:40It would mean that there is some link with humans and brain health. So what is K -L -V -S that you mentioned and what is it associate with? As we've discussed, we all have cloth O circulating in our blood and around our brain. But some of us, about one to four, one to five of us, will carry a gene for cloth, though a genetic code for cloth, though, that leads to higher levels of production. And KLVS refers to two single nucleotide polymorphisms that cause a difference in the coding of the protein itself. Again, so about one to four, one to five of us will have KLVS, other people are non -carriers, and those people with KLVS will genetically have higher levels of cloth, those circulating in their blood.

1:31:38Now, interestingly, it's only the people with one copy of KLVS, the heterozygates, and very rarely there are homozygates of KLVS, and those people end up having multiple disadvantages, and life span, and vulnerability to different diseases. So when we talk about KLVNs, we're talking specifically about heterosigacity, caring one, a leal of it. What are some of the health consequences of being homozygous, and what is the prevalence of homozygacity? It's really rare. So if the prevalence of heterosigacity is around 25 % in populations, homozygacity is so... It's probably about one to two percent because actually it would kind of mirror apoe4 where the heterozygosity is about the same.

1:32:33It's about one in four and the homozygosity is about one in one in 50 to one in a hundred. Yeah, that's about right. It's pretty rare, but it exists and those people have shorter lifespans. They have much lower levels of clotho and they are at risk for anything that the heterozygosity helps with, the homozygosity hurts. But the heterozygosity has been a really interesting window into cloth -o and natural experiments. How much higher is their cloth -o level? Well, we did this study. Jennifer and I took the serum from individuals here at the Memorane Aging Center a few hundred and tested the ones that were non -carriers that just have the typical clothogene versus the KLVS heterozygate carriers.

1:33:26And the heterozygate carrier status increased clothilevels by about 15%, 15 maybe 20%. And just to give you context, if you're wondering, am I carrier and some people are and some people aren't, I don't want people to forget that exercise is thus far one of the more powerful modulators of clotheo -expression and exercise increases clotheo on average by 30 % so much more than with the genetic influences. But nonetheless, KLVS increases clotheo levels by about 15%. So for example, if an average number let's say a non -carrier had 800 pg per mill of clotheo circulating in their serum. A KLVS carrier might have like 950 or so pica grams per mill.

1:34:19That's what I'm remembering from our graphs. There's reason to believe that it increases cloth though because those two amino acid changes that translate into a different amino acid in the protein itself influences is a creation of cloth though from cells. There's a biologic reason that this variant is probably changing cloth levels. The other piece to this story, the one that's just so fascinating, is now when you take those data, the prevalence of heterozygosity of KLVS, and you cross it with APOE4. So now we have listeners of this podcaster absolutely not strangers to the population -based risk of APOE4.

1:35:07Of course, we always want to remind people that at the individual level, very difficult to make that statement. But at the population -based level, we know that having an APOE3 and an APOE4 is at least a doubling of risk, maybe even slightly more, and homozygosity for APOE4 could be an eight to 12 fold increase, so call it a log order increase in risk. So tell me, what did you find in people who are homozygous and heterozygous for APOE4, who also happen to be heterozygous, i .e. have the favorable variant of KLVS, single copy? This is really remarkable data. I first want to go back to that original experiment that we published in 2014, I believe, with Jennifer, and that those people that carried the KLBS allele did better across cognitive testing.

1:36:03And this is a normal aging. This was not an Alzheimer's disease, but carrying that variant, carrying KLBS associated with better cognition across the board. I'm often asked, well, how much smarter were those individuals? And I would be careful about smarter and happier, and we don't know, but the cognitive tests showed us that they did better and they did better to the same extent that apoe for carriers would do worse. So as a sort of a similar amount of change in cognition in which KLBS was improving and apoe for was decreasing, with that in the background since then, many groups and Many people have looked at KLBS in their populations and that association has held largely in most studies, not in all, but in most studies, there is an association of KLBS and better cognition with heterosigacity.

1:37:05So then the question comes that you asked about APOE4, Alzheimer's risk and clothO in KLBS. We initially did a study in collaboration, actually led by Ozeomo Concoo and his group at Wisconsin, that showed that in individuals with ABOE4 that were carriers of KLBS, that they just had less of the effects of ABOE4 in terms of less abated deposition, less cognitive problems. It was a smaller study of a few hundred people at risk for Alzheimer's disease, but it was a good study. But I'll tell you the study that I'm most excited about, that we were not a part of. That came from Stanford, led by Mikhail Beloy.

1:37:55He's one to watch. He just established a lab at WashU and his senior PI, Michael Gritius, and their wizards at doing genetic population studies. So Peter, they did this remarkable study of 22 different cohorts that were normal cognition, MCI, mild cognitive impairment that you mentioned earlier in Alzheimer's disease, over 20 ,000 individuals, a very large meta -analysis, and they looked at if there was a relationship between KLVS carriers and ApoE4. And the bottom line of what they found was that if an individual carried KLVS, the ApoE4 didn't matter. So let me break down with that actually meant in their experiment.

1:38:46That meant that in those people that carry ABOE4, if they were also heterozygate for KLVS, they had a decreased risk for developing Alzheimer's disease that was pretty close to the normal population. They had a decreased conversion from MCI to AD, and they had decreased Alzheimer's biomarkers, both in their CSF and in their brain. They just had less amyloid beta. It's a really striking study. Again, because it has the power of statistical analysis, it holds across many, many cohorts and it's many, many people and KLVS essentially blocks and abrogates the apoi for toxicity by this genetic associations really remarkable.

1:39:39Did the double E -forced, the homozygates reduced to a completely normal three -three risk? My recollection, but it's been a while since I looked, there's a graph that demonstrates all of this that's a great summary. My recollection, which could be wrong, that's why I'm asking, was that the three fours were completely abrogated to a three -three risk, and that the four fours ended up coming way down, but they still looked like more of a three -four. Am I misremembering that? They didn't actually publish that. They excluded the homozygates from the analysis, but it might be an assoupliment somewhere, but I actually reached out to Mikael and I asked him about the 4 -4.

1:40:23I had the same question and so this is just by verbal communication, he indicated that the 4 -4 also associated with the protection from the KLBS heterosigacity, so that the effect of having one able E4 wasn't different than having two able E4s in terms of KLBS protection. It protected in both heterosigacity and homosigacity. of the April, April, April. Again, it's just through verbal communication. It might have been an supplement somewhere. So did they find anything negative? So going way back to the mechanism of this, which at least in part is communicated through a platelet factor, was there anything that might have been unwarranted such as an increase in stroke risk?

1:41:11I mean, I know that it's actually the opposite. But I'm just trying to say, like, is there anything that with an increase in a platelet factor that could have led to an increase in DVTs or something. I mean, when you have a sample size as large as they did, presumably you would find something that was negatively associated with the KLVS. I'm not aware of increase, as you were saying, it's actually the opposite. The KLVS heterosigacity actually associates with the protection against stroke risks, cardiovascular risks. Even metabolic risk. metabolic, yeah, metabolic diseases. I searched for something that could be negative.

1:41:52And I did find that in the cancer literature, KLBS heterozygosity has a poor prognostic, indicates a poor prognosis in BRCA1 carriers with breast cancer. There are examples where it's not helpful. And one is in BRC and one positive breast cancer. Dean, going back to the human homozygots for KLVS, do we know how much of an increase in cloth though they produce? So if the heterozygots are producing 15 to maybe 20 % more, how much more cloth though is being produced by the homozygots? And does that give us an insight into toxicity in humans that was not observed in mice and not even observed in the primates?

1:42:44Because in the primates, when you gave too much, you just didn't get a benefit, but you didn't get a harm in the mice. You actually got more and more benefit is the organisms get more and more complicated. It seems that the therapeutic windows getting narrower and narrower to the homozygots give us any insight into that. With Jen here at the Memorane Aging Center, we have access to homozygate serum, and we actually looked at several homozygates compared to non -carriers and heterozygates. And what we found is that their cloth o levels are actually lower than normal. And we think that has something to do with, I'm going to get a little deeper into this, because I think it's really important when we think about therapeutic, actually, that But many genetic variants won't cause a change in the protein sequence itself.

1:43:34If a nucleotide change is in an intron, it's just not going to change the structure or function or anything of the protein. But in KLVS, there are two nucleotide changes, and they translate into different amino acids and two places of the clothoprotein. And so what we think is happening, this is speculation, but what we think is happening based on in vitro studies that Arcing did in 2002, that in the clotho -heterosigate in which one allele is making a mutant protein, we think that there's an overcompensation for that mutant protein by increasing wild -type levels. Because what that variant does is it mucks up clothose secretion from the cell.

1:44:27So just to be clear, carrying one variant is likely changing the structure and function of clotho, but there's probably an overcompensation from the wild type allele, causing higher levels of the wild type clotho. First of all, that is an unbelievable. I would not have guessed that, and I can't believe I didn't think to ask such an obvious question, which is do the KL VS Individuals produce the same protein? I took it for granted that they produced the same protein and just made more of it but to be clear if you As say those individuals are you finding two proteins in them a The protein that mirrors the wild type, that's the one that's elevated 15 to 20 percent, and an actual mutated protein or a protein that has two different amino acids, which, again, to many people sounds like big deal.

1:45:24You've got 1 ,000 amino acids in the full protein. You've changed two of them. How much can it matter? Well, unfortunately in biology, it can always matter, ask a patient with sickle selenemia. So is that what you're seeing? you're seeing two different proteins and it's the wild type one that is over -expressed. At this point, it's the hypothesis that the wild type one is really compensating, overcompensating by increasing levels for what may be a mutant protein. But in reality, and we've done aliza's on the enzyme linked amino acids on thousands and thousands of individuals, but the Eliza itself doesn't distinguish between a KLBS protein and a normal wild type clothoprotein.

1:46:11It doesn't distinguish between the two. And so the answer to that question is not known. Eliza is just too blunt an instrument to figure that out. We take that a step further and say, what's happening in a clothopomazigate? They're expressing a mutant protein from one allele that mux -upsecretion, and they're expressing a mutant protein from another allele that mux -upsecretion, and so they don't have wild -type clotho. All of their clotho is the KLBS form, and it's very low. How much lower is it, Dina? In our studies, it was, I'm trying to visualize the graph. If there is a 15 % increase with heterosigacity compared to non -carriers, there is probably a 30 % decrease with homosigacity compared to non -carriers.

1:47:04So, to give you a sense of numbers, if a non -carrier was at 800, a KLVS homosigate carrier was below 600, something like that. This is why this matters. It matters for many reasons, but there have been some really large -scale studies recently, Peter. One is called the N -Hain Study Published in 2022, I believe. And what this study showed us is that clothal levels, as measured by this immuno -assay, by Eliza, that the clothal levels really correlated with mortality. I want to make sure I get this right. So it was over 10 ,000 people in the United States. Clothal serum was measured on everyone.

1:47:51The mean age was about 56 years. And if the meanclothal level was around 800, then something less than what they defined as 666 by their study, Pika Grams for Mill, associated with a 30 % mortality over five years. and that was replicated in another study called in Chianti, that lower levels of cloth though. And again, this isn't like half the levels. This is maybe it's like 30 % of a decrease of levels associated with a 30 % mortality over five years. And that mortality was primarily in cancer and cardiovascular disease. I think levels are gonna matter. As we think about our human aging, as we think about our organismal health, heart health, brain health, cancer health, the levels at least by association right now matter, and lower levels are really associated with more diseases of aging.

1:48:52It's just like, can we even scratch the surface of this? I mean, we've just sat here and talked for two plus hours about the limits of our understanding of cloth, though, and brain health, and yet this end -hane study is actually looking at something totally different, which is all cause mortality and saying basically if you're in your 50s and your clotho levels are 30 % below what would be expected for somebody or age, it's associated with a 30 % increase in all cause mortality over the next five years. If that turns out to be causal and you're saying that the manner in which that death was distributed was cardiovascular and cancer.

1:49:33So it doesn't even have to do with more dementia, which of course wouldn't be kicking in in your fifties. It suggests that Clotho is doing even more than just protecting brain health, which if it did nothing else would still be arguably the most important thing that we should be trying to get into humans for clinical trials. So I know that your area of expertise is not on college or cardiovascular medicine, but I'm just curious as to what your thoughts are as to how that can be happening. I couldn't agree with you more and my expertise is as a neurologist and neuroscientist is really on the brain, but having said that, the side effects of clotho increasing it may be much more than in helping brain health.

1:50:17It clearly has a very strong association with protection against cancer, cardiovascular disease, and kidney disease. There is Peter, a very, very, very large literature on clotho and organ health, a very strong one. In the cancer field, there are many preclinical models, again in rats and mice that show that giving clotho, the soluble hormonal form actually stopped and reversed cancers and mice, like pancreatic cancer, for example. And then there are association studies in humans too, but there is so much more to the biology of clotheau than only what it's doing in the brain. In kidney disease, clotheau is going to come to market and maybe the kidney people beat the brain people.

1:51:09If clotheau comes to market, I think people are going to benefit no matter what. But in chronic kidney disease, there is a decrease in clotheau, secretion and decrease in clotheau in the blood. And the kidney folks, the kidney specialists are really developing Clotho as a diagnostic biomarker to understand when the kidney starts failing. The idea there is that right now the measures are glomerular filtration rate, urea, and these are not as sensitive as they would want biomarkers to be in detecting kidney dysfunction. As soon as the kidney starts having trouble, clotho declines as soon as it starts having trouble.

1:51:54So there's a fervent interest in developing clotho as a biomarker for kidney function. And I would say, again, if we think in a broader sense about clotho, maybe someday, and maybe someday soon, we would have our clotho levels checked, just as we do our blood pressure and our cholesterol, and we get breast exams, and we have colonoscopy. why not have a cloth a level checked? Everyone's will be different. Now, how do we get around the issue that it has this diurnal effect? That's the only thing that jumps to my mind when you're talking about a marker of EGFR because I know in our practice, we're fanatical about monitoring EGFR.

1:52:36I mean, we look at Cistatincy, we look at creatinine, we're triangulating them, at least to our ability. These don't depend terribly on time of day. So do you think that the answer for cloth though is you have to have it done 60 minutes after you wake up in the morning It's non -negotiable. Don't ever let somebody check your cloth at level at two in the afternoon. Otherwise, we can't interpret it Is that how you think it's going to factor in? I think that is going to give us the best and more accurate level of cloth though And in fact, I mentioned that we've done thousands and thousands of cloth patho measurements and human serum and human CSF through those final fluid.

1:53:16And I always make sure that whoever we're collaborating with and who has collected the specimen did it in a morning fasting manner so that the individual or patient hasn't eaten and it's first thing in the morning. Why do you know that eating impacts Hotho levels? I don't know that and I don't know that it's really been tested per se, but in the spirit of what one does, for example, just being incredibly consistent and rigorous in a way with less confounding factors in the background. But having said that, the decline over the course of a day isn't dramatic. The decline by age is more relevant than the decline by time of day.

1:54:01I would say that the decline by age is a consistent decline. But the time of day does matter because it can decrease by almost 40 % by midnight. And this is done with I have to say, I've looked through the literature, the diurnal rhythm of cloth has been shown in a small number of people with the data look good, that it's not really statistically different until like afternoon, but it's starting to decline. I had to design the time and day and I would say that we need a cloth that people take in the morning, fasting like they do with their cholesterol, just get it along with the cholesterol, draw.

1:54:42And most importantly, we need a standardized assay. Yeah, it's about to say, is there a clear -based assay for this? No. No, I think it's coming given how many people and how many samples levels haven't been coordinated between institutions and labs. labs. So everything we do in our lab is consistent to our methods and standards and we standardize everything to, and I include my own samples in there, but it's all standardized. But then my lab may have slightly different values than what is going on in another lab. And then of course, so if I woke up in the morning and I checked my level and I was at 800, is it pkg per milliliter?

1:55:23Is that the unit? Yeah. Yeah. If I did my test at six o 'clock in the morning and I'm 800 pg per milliliter and then from seven o 'clock to nine o 'clock in the morning, I'm in the gym. I could easily be 1200 pg per milliliter after. So how does that impact our understanding of what's going on? I think that's a really good important point. I do want to emphasize that the human studies show chronic exercise increases cloth o by after a few months after three months. And there's been a mouse study that shows an acute bout of exercise can really nearly double cloth though. But I think we would be interested again.

1:56:01We're imagining and not so distant world. It would be important to get a baseline level. Where are you living? So just to be clear, you're saying you do not have human data that demonstrate that acute bouts of exercise dramatically changes. I have not seen that. I have not seen that. I know a person and who might be willing to do that experiment for you. We could discuss that offline. Sure. Okay, so final question on this, Tina, because I know it's come up for us a lot in our practice. So I'm hoping you have a better solution for us. We do like to test our patients, especially those who are higher risk for Alzheimer's disease.

1:56:44We do like to test to see if they have the KLVS polymorphisms. It is very difficult to get that information. Are you guys doing that work yourselves? Are you outsourcing that? How are you guys getting that information on your own? I mean, I realize that much of the research you're doing is on a database where that's been done. But are you aware of any commercially easy ways for individuals to determine their KLVS status? Should they choose to know? I don't know the exact answer to this. I know that the information is represented somewhere on 23 and me. We can't seem to get it out of 23 and me even when we use Prometheus.

1:57:25Yeah, we can't seem to do it. Hopefully somebody listening can say, no, you're wrong Peter, you just got to do this, but we haven't figured it out. Yeah, I have to say that in our case, when we collaborate with the clinical researchers, they have their populations completely genotyped or cloppoeple 4 and most other genes out there with polymorphisms. There's been a really deep sequencing and genetic screen of all those individuals, but I'll have to circle back to you about that. Okay. Well, this has been, I mean, I think at the risk of being hyperbolic, just an insanely fascinating discussion.

1:58:04And I'm so happy to be able to have discussed your work with you. And then by extension to be able to put this in front of so many people, because I do think that for a disease like Alzheimer's disease that if we're being brutally honest of all the what I call the forehorsement of death, if there's one that we have very, very little to offer patients, once they're in the throes of the disease, this is the sad poster child for it. It is unfortunately today. I don't need to tell you what you see every day, but I think people watching this understand that even with the advent of anti -Maloid therapies, they have barely, barely been able to put a dent in this enormous problem.

1:58:46It's for that reason that I'm very excited about this and just applaud you on your career. I think this is only the beginning. We have to get this into human clinical trials. That's what's next. Thank you so much for this invitation. It's been really, really fun talking to you. Alzheimer's disease is one of our biggest biomedical challenges. Our entire world is aging. It used to be the US, Japan, Sweden, but it's the entire world. China, India, Africa, all continents are aging. We're aging rapidly. Again, this is one of our biggest biomedical problems. we really do not have effective therapies.

1:59:28And I'm hopeful that with multiple shots on goal right now, something will come to market that provides that resilience. In addition to anti -emaloid therapies, that is a cocktail that provides resilience for the suffering that really erodes our memory, it destroys families, economies, it's just a devastating problem. We're glad to be working on it, and we hope that we can do something big and important, but that remains to be seen. I also just wanted to say I'm so lucky to be doing what I love to do, really, really love to do. And I am also lucky enough to have a very diverse portfolio of being funded by the NIH, by foundations, and in the past five biotech, and also by philanthropy.

2:00:17be. I can't emphasize enough how much friends and supporters of our lab have enabled really risky science, have enabled us to just take a leap, ask big questions, take a big risk, and see what happens. And when or lose to really see what happens when we go down that route, and I think that's so important to progress in science is to do something risky to take a risk. So I'm very grateful to be doing what I'm doing. I'm glad you mentioned that I do think that philanthropy can have an enormous impact in science when philanthropists come in with the right attitude, which is, I want to fund something that is asymmetric in the following way.

2:01:04It is too risky for, say, the NIH to fund or too risky for industry to come in and fund, but yet it has enough biological plausibility and enough potential upside that if it works, it's a game changer. And look, the reality of it is, I think Clotho is a poster child for that type of work. And at least as of this moment, it looks like it's actually paying off. So I and countless others are grateful for what's been done. Dina, thank you so much for your time today. Really, really appreciate it. Dina, thank you. This is wonderful. Thanks. Thank you for listening to this week's episode of The Drive.

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